Marker molecules associated with lung tumors

ABSTRACT

The present invention relates to nucleic acids and polypeptides associated with lung cancer. The invention is more specifically related to a nucleic acids and the polypeptides transcribed thereof, the expression of which is significantly altered in association with lung cancer. The invention relates to a series of differentially spliced transcripts of the gene disclosed herein, that are associated with tumors of the respiratory tract. Furthermore the present invention provides a method for early diagnosis, prognosis and monitoring of the disease course and for therapy and vaccination of cell proliferative disorders such as e.g. lung tumors.

This application is a divisional application of U.S. application Ser.No. 10/515,477, filed Nov. 19, 2004, which has issued as U.S. Pat. No.7,355,025; which is a National Stage of International ApplicationPCT/EP03/50175, filed May 16, 2003, published Nov. 27, 2003, under PCTArticle 21(2) in English; which claims the priority of EP 02010275.2,filed May 21, 2002. The above-identified applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to nucleic acids and polypeptidesassociated with lung cancer. The invention is more specifically relatedto a nucleic acids and the polypeptides transcribed thereof, theexpression of which is significantly altered in association with lungcancer. The invention relates to a series of differentially splicedtranscripts of the gene disclosed herein, that are associated withtumors of the respiratory tract. Furthermore the present inventionprovides a method for early diagnosis, prognosis and monitoring of thedisease course and for therapy and vaccination of cell proliferativedisorders such as e.g. lung tumors.

BACKGROUND OF THE INVENTION

In the developed countries death rates from cancer declined throughoutthe past decade. One exception from this decline in cancer caused deathrates is lung cancer. Overall lung cancer is one of the few cancers inthe world still showing increasing incidence. Lung cancer is the leadingcause of cancer deaths in both men in women. Moreover the survival ratein lung cancer is poor up to now and despite the scientific and medicalefforts in the field of lung cancer there was hardly any increase in thesurvival rate.

In lung tumors as in most other tumors, there is a strong correlationbetween the patients' outcome following initial therapy and the stage atwhich the disease has been diagnosed. So the earlier the cancer could bedetected, the better are the chances for the patient to survive. Thussensitive testing methods are required for detecting the tumors in earlystages.

The most promising methods for early diagnosis of tumors are thoseinvolving molecular markers characteristic for tumor cells.

Lung cancer is a quite heterogeneous disease. Multiple regulators of thecell growth can be involved in the genesis of cancer. These regulatoryelements of the cell cycle can be either positive regulators, namedoncogenes when mutated, so that a transformed state is reached, ornegative regulators, named tumor suppressor genes. The number of factorsknown to be involved in the regulation of the cell cycle and potentiallybeing candidates for the development of cancer exceeds 100 up to knowand is still increasing.

The molecules being involved in the emergence of the cancerous state ofa cell can be used to discriminate between cancer cells and normaltissue. Thus cancerous tissue can be detected by detecting moleculescharacteristic for the cancer cells. This turns out to be sophisticateddue to the large number of molecules potentially being involved incausing cancer.

For improved diagnosis of tumors, there is a need for new markermolecules for use in diagnosis of cancers and especially of lung cancer,which enable for specific early detection and give the opportunity totreat the disorders at an early stage.

SUMMARY OF THE INVENTION

The present invention provides nucleic acids and polypeptides associatedwith lung cancer. According to the present invention these molecules maybe used as molecular markers that allow for comprehensive detection ofcell proliferative disorders such as e.g. lung tumors even at earlystages.

The present invention thus provides polypeptides and nucleic acids, theexpression of which is significantly altered associated with lungtumors, which allow for enhanced prognosis and diagnosis of diseasesassociated with abnormalities of the growth of cells. Furthermore thenucleic acids disclosed herein comprise transcripts arising fromalternative splicing of genes, that do not occur in normal tissue in theextent they may be found in tumorous tissue.

In another aspect of the invention, the nucleic acids and/orpolypeptides disclosed herein alone or in combination with othermolecules may be used for therapy and/or vaccination of cellproliferative disorders such as e.g. lung tumors.

Yet another aspect of the present invention are pharmaceuticalcompositions containing polypeptides and/or polynucleotides disclosedherein alone or combination with one or more other therapeutic ordiagnostic agents and/or carrier or adjuvant substances.

The present invention also provides kits such as diagnostic kits orresearch kits for the detection of the polynucleotides or polypeptidesdisclosed herein or comprising the polynucleotides or polypeptidesdisclosed herein or combinations thereof.

During the experiments leading to the present invention a gene wasidentified, the expression of which is associated with lung tumors. Thepresent invention furthermore is based on the inventors findings shownin Examples 1 to 6, that the level of expression of nucleic acids aswell as of polypeptides transcribed from the marker gene presentedherein in FIG. 1-11 in samples allows to diagnose and grade cellproliferative disorders such as e.g. lung tumors, to predict the courseof the disease and to follow up the disease after initial therapy.

The present invention thus provides novel nucleic acids and polypeptidesassociated with lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts human LUMA1 mRNA variant 1 sequence (SEQ ID NO: 1) andthe encoded LUMA1-protein isoform 1 Sequence (SEQ ID NO: 2).

FIG. 2 depicts human LUMA1 mRNA variant 2 sequence (SEQ ID NO: 3).Differential splicing of exon 7 leads to deletion of part of exon 7which introduces a frameshift in the 3′ sequence of the splice variant.Compared to mRNA variant 1, the encoded protein in variant 2 (SEQ ID NO:4) is shorter than the one in mRNA variant 1. The frameshift leads to aprotein isoform with a different carboxy terminal sequence. In additionthe complete exon can be spliced out (not shown).

FIG. 3 depicts human LUMA1 mRNA variant 3 sequence (SEQ ID NOs: 5 and6). This variant is characterized by a 3 bp deletion and a differentpolyadenylation site which leads to a shorter 3′ sequence.

FIG. 4 depicts human LUMA1 mRNA variant 4 sequence (SEQ NOs: 7 and 8).This variant is characterized by a deletion of exon 3.

FIG. 5 depicts human LUMA1 mRNA variant 5 sequence (SEQ ID NOs: 9 and10). This variant is characterized by a deletion of exon 3 and exon 4.

FIG. 6 depicts human LUMA1 mRNA variant 6 sequence (SEQ ID NOs: 11 and12). This variant is characterized by a deletion of exon 3, 4 and 5.

FIG. 7A depicts human LUMA1 mRNA variant 7 sequence (SEQ ID NO: 13).FIG. 7B depicts protein 1 sequence (SEQ ID NO: 14). Protein 1 is encodedby an ORF located at the 5′-end of the mRNA.

FIG. 8A depicts human LUMA1 mRNA variant 8 sequence (SEQ ID NO: 15).FIG. 8B depicts protein 2 sequence (SEQ ID NO: 16). Protein 2 is encodedby an ORF beginning at base pair 2242. The open reading frame isterminated by a stop codon at bp 2863-65. This stop codon is absent inmRNA variant 9 encoding protein 3 (see FIG. 9).

FIG. 9A depicts human LUMA1 mRNA variant 9 sequence (SEQ ID NO: 17).FIG. 9B depicts protein 3 sequence (SEQ ID NO: 18). The N-terminal partof protein 3 is present in protein 2. Due to the absence of the stopcodon at bp 2863-65, the open reading frame extends to bp 4290.

FIG. 10A depicts human LUMA1 mRNA variant 10 sequence (SEQ ID NO: 19).FIG. 9B depicts protein 4 sequence (SEQ ID NO: 20). Protein 4 is encodedby an ORF starting at bp 2866 due to the presence of a 5′ stop codon atbp 2863-65. Protein 3 (FIG. 9) and protein 4 do harbour a differentc-terminus compared to the protein depicted in FIG. 1 because of adifferential splicing at exon 8 (exon T) which extends the exon to the5′ end leading to a frameshift.

FIG. 11 depicts genomic sequence of the human LUMA1 gene (SEQ ID NO:21). LUMA1 exon sequences are underlined. The genomic sequence harborsLUMA1 exon 1 to exon 8. Two different splice acceptor sites aredetectable at exon 3 leading to an additional codon which is indicatedby (>). Exon 3 or exons 3 and 4 or exons 3, 4 and 5 are differentiallyspliced. In addition differential splicing of part of exon 7 has beendetected. Whereas exon 3, exons 3 and 4, and exons 3, 4 and 5 deletionslead to an in frame deletion of internal LUMA1 amino acid sequences thedifferential splicing of part of exon 7 generates a frameshift. Thisframeshift generates to different carboxy terminal LUMA1 proteinisoforms. The two splice acceptor sites at exon 7 are indicated by (>).In addition two different polyadenylation sites of the LUMA1 gene areindicated.

FIG. 12 depicts mouse LUMA1 mRNA sequence (SEQ ID NOs: 22 and 23).

FIG. 13 shows nucleotide sequence comparison of human (nucleotides 1 to1160 of SEQ ID NO: 1) and mouse LUMA1 (nucleotides 28 to 1185 of SEQ IDNO: 22) mRNA. Over the entire coding sequence, a strong sequenceconservation during evolution is detectable.

FIG. 14 shows nucleotide sequence comparison of human and mouse LUMA1mRNA (nucleotides 1 to 1160 of SEQ ID NO: 1 and nucleotides 28 to 1185of SEQ ID NO: 22). Sequence blocks which are identical in human andmouse LUMA1 are boxed.

FIG. 15 shows amino acid sequence comparison of human and mouse LUMA1protein. Sequence blocks which are identical in human (amino acids 1 to391 of SEQW ID NO: 2) and mouse (amino acids 8 to 392 of SEQ ID NO: 23)LUMA1 are boxed. Highly conserved peptide sequences are presentindicative for a highly conserved function during evolution.

FIG. 16-20 shows detection of the expression of LUMA1 in lungadenocarcinomas (FIG. 16: NI/TI; FIG. 17: N2/T2; FIG. 18: N3/T3; FIG.19: N4/T4; FIG. 20: N5/T5) using the real time PCR technique (ABI TaqMan7700). For amplification of the LUMA1 gene the following primers wereused: LUMA1-A: CTCGTCAGGCGACCTTATATC (SEQ ID NO: 24); LUMA1-B:TGTCAGTTGAACATTTTCTGCC (SEQ ID NO: 25). For the analysis correspondingtumor and normal samples were used.

FIG. 21 shows gel electrophoresis of the endpoint PCR of theamplification of the LUMA1 transcript in lung adenocarcinomas andcorresponding normal tissue. In tumor T1 and T3 enhanced expression ofLUMA1 was seen, in tumor T4 and T5 a strong overexpression of LUMA1compared to the corresponding normal sample. The PCR products analysedon the agarose gel, were derived from the real time PCR reactions shownin FIG. 16-20.

FIG. 22 shows gel electrophoresis of the endpoint PCR of theamplification of the LUMA1 transcript in lung adenocarcinomas andcorresponding normal tissue. Whereas in normal sample N1, N3 and N4 noLUMA1 transcript was detectable, in lung adenocarcinoma T1 and T3expression of LUMA1 transcript was detected. In T4 no amplification wasvisible. In T2 and T5 an enhanced expression was detected. Foramplification of the LUMA1 gene the following primers were used:LUMA1-C: CTCGTCAGGCGATACTCCC (SEQ ID NO: 26); LUMA1-D:CACCAGTCAGCTCTAAATGGG (SEQ ID NO: 27).

FIG. 23 shows gel electrophoresis of the endpoint PCR of theamplification of the two different LUMA1 exon 7 (exon S) transcripts innormal tissues. 11 normal tissue samples were tested for expression ofthe long variant of exon 7. Only in testis and liver an expression ofthis exon 7 splice variant has been detected. In addition, theexpression of the short exon 7 splice variant could be observed intestis, liver and stomach. NTC=no template control.

FIG. 24B shows immunohistochemical analysis of lung adenocarcinoma, andFIG. 24A shows that corresponding normal tissue employing a primaryantibody directed against LUMA exon 2. In the carcinoma tissue and inthe corresponding normal lung tissue a cytoplasmic staining has beendetected with the polyclonal antibody directed protein sequences of exon2. No difference in the staining pattern between normal and tumor hasbeen observed.

FIG. 25B shows immunohistochemical analysis of lung adenocarcinoma, andFIG. 25A shows the corresponding normal tissue employing a monoclonalprimary antibody directed against LUMA exon 7. In the carcinoma of thepresented sample a strong overexpression of the luma exon 7 (longtranscript) protein isoform is detectable. The tumor cells show acytoplasmic staining pattern. In the corresponding normal lung tissue nostaining has been detected with the exon 7 specific monclonal antibody.

FIG. 26A shows immunohistochemical analysis of lung carcinoid, and FIG.26B shows squamous cell carcinoma employing a monoclonal antibodydirected against LUMA exon 7. In the carcinoid (A) and in the squamouscell carcinoma (B) a strong overexpression of the LUMA exon 7 (longtranscript) protein isoform has been detected. The tumor cells show acytoplasmic staining pattern.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the method according to the present invention isespecially useful for early detection of cell proliferative disorderssuch as e.g. lung tumors and for detection of disseminated tumor cellsin the course of diagnosis of minimal residual disease.

In another aspect of the present invention, the nucleic acids orpolypeptides disclosed herein may be used in the course of diagnosis ofdisorders associated with abnormal proliferation of cells in samplessuch as tumor resections, biopsies or the like. In this aspect theinvention provides a method, which allows to build a strategy for thetherapy of diseases according to their molecular properties. Accordingto the present invention, the level of said polypeptides and/or nucleicacids can be used as a molecular marker for prognosis, monitoring andthe design of a strategy of tumor therapeutics.

It is yet another aspect of the present invention to provide methods foridentification of molecules binding to the nucleic acids andpolypeptides of the present invention as well as of activators andinhibitors of the expression of the genes of the present invention. Alsoa method for the identification of drug candidates for the therapy ofproliferative disorders is provided.

The present invention provides tumor associated nucleic acids andpolypeptides characterized by the sequences given in FIG. 1-11.

Marker molecules as used in the present invention my comprise nucleicacids and polynucleotides. On the level of nucleic acids the markermolecules may be DNA or RNA comprising genomic DNA, cDNA, and RNA suchas mRNA or hnRNA. In one preferred embodiment of the invention, nucleicacids arising from particular differential splicing events may be markermolecules.

Expression as used according to the present invention may comprise forexample expression of proteins. The transcription to RNA and thus thelevel of mRNA may also be understood to be expression according to thepresent invention.

The expression of a compound is said to be significantly alteredaccording to the present invention, if the level of expression differsby more than 30%. The alteration of the expression may comprise forexample elevated expression or reduced expression of said compound.Another aspect of the altered expression may be an alteration in a way,that the compound is expressed under non wild-type circumstances. Thismay comprise, that the compound is for example expressed in situationsthat naturally suppress the expression, or is not expressed insituations that naturally induce the expression of the compound.

Alteration of the expression as used herein may also comprise analteration in the transcription pattern of a gene. E.g. the alterationof the transcription pattern may comprise alternative splicing of thegene. The alterations in the transcription pattern may influence thepolypeptides translated from the altered transcripts or may berestricted to untranslated regions. The alteration in the transcriptionpattern of a gene may comprise use of novel exons in the transcripts,deletions of exons in the transcripts or the variation in the ratios ofdifferent splicing variants in cells. Thus alterations intranscriptional patterns of genes as used herein may comprise theproduction of nucleic acids such as e.g. mRNA, cDNA etc. containingadditional stretches of nucleic acid sequences compared to wild typenucleic acids occurring in control tissues. Alternatively the nucleicacids produced by alternative splicing patterns may produce nucleicacids missing stretches of nucleic acid sequences present in wild typepolynucleotides. The presence of additional stretches may occursimultaneously with the absence of original sequence-stretches in singletranscripts. Alterations in the expression of genes as used in thecontext of the present invention may also comprise an alteration in thelevel of expression of splicing variants of genes. This may includeincreased or decreased expression of particular splicing variants aswell as expression of variants not present in wild type tissue or theabsence of expression of splicing variants present in wild type tissue.In one embodiment, the alteration of the expression of the splicingvariants may comprise the alteration of the ratios of different splicingvariants in said tissue.

Nucleic acids as used in the context of the present invention arepreferably polynucleotides or fragments thereof. Preferredpolynucleotides comprise at least 20 consecutive nucleotides, preferablyat least 30 consecutive nucleotides and more preferably at least 45consecutive nucleotides, which are identical, share sequence homology orencode for identical, or homologous polypeptides, compared to thepolypeptides associated with the proliferative disorders disclosedherein. The nucleic acids according to the present invention may also becomplementary to any of said polynucleotides. Polynucleotides may forexample include single-stranded (sense or antisense) or double-strandedmolecules, and may be DNA (genomic, cDNA or synthetic) or RNA. RNAmolecules comprise as well hnRNA (containing introns) as mRNA (notcontaining introns). According to the present invention thepolynucleotides may also be linked to any other molecules, such assupport materials or detection marker molecules, and may, but need not,contain additional coding or non-coding sequences.

The polynucleotides according to the present invention may be nativesequences or variants thereof. The variants may contain one or moresubstitutions, additions, deletions and/or insertions such that theimmunogenicity of the encoded polypeptide is not diminished, relative tothe respective native proteins. The variants show preferably 70%, morepreferably at least 80% and most preferably at least 90% of sequenceidentity to the native nucleic acid molecules used in the methodsaccording to the present invention. Methods for determination ofsequence similarity are known to those of skill in the art.

One example for detecting the similarity of sequences can be carried outusing the FastA and/or BlastN bioinformatics software accessible on theHUSAR server of the DKFZ Heidelberg.

Furthermore nucleic acids according to the present invention are allpolynucleotides, which hybridise to probes specific for the sequencesdisclosed herein under stringent conditions. Stringent conditionsapplied for the hybridisation reaction are known to those of ordinaryskill in the art and may be applied as described in Sambrook et al.Molecular cloning: A Laboratory Manual, 2^(nd) Edition, 1989.

The present invention also provides polynucleotides, which due to thedegeneracy of the genetic code encode the polypeptides natively encodedby the disclosed nucleic acids while not showing the percentage ofsequence homology as described above within the nucleic acid sequence.Such nucleic acids might arise by changing the codons present in thedisclosed sequences by degenerate codons and so preparing a syntheticnucleic acid.

The nucleotide sequences according to the present invention may bejoined to a variety of other nucleic acid sequences using the knownrecombinant DNA techniques. The sequences may for example be cloned intoany of a variety of cloning vectors, such as plasmids, phagemids, lambdaphage derivatives and cosmids. Furthermore vectors such as expressionvectors, replication vectors, probe generation vectors and sequencingvectors may be joined with the sequences disclosed herein. Sequences ofspecial interest, that could be cloned to the nucleic acids according tothe present invention are for example non coding sequences andregulatory sequences including promoters, enhancers and terminators.

In a preferred embodiment, polynucleotides may be formulated such thatthey are able to enter mammalian cells and to be expressed in saidcells. Such formulations are especially useful for therapeutic purposes.The expression of nucleic acid sequences in target cells may be achievedby any method known to those skilled in the art. The nucleic acids mayfor example be joined to elements that are apt to enable theirexpression in a host cell. Such elements may comprise promoters orenhancers, such as CMV-, SV40-, RSV-, metallothionein I- orpolyhedrin-promotors respectively CMV- or SV40-enhancers. Possiblemethods for the expression are for example incorporation of thepolynucleotides into a viral vector including adenovirus,adeno-associated virus, retrovirus, vaccinia virus or pox virus. Viralvectors for the purpose of expression of nucleic acids in mammalian hostcells may comprise pcDNA3, pMSX, PKCR, PEFBOS, cDM8, pCEV4 etc. Thesetechniques are known to those skilled in the art.

Other formulations for administration in therapeutic purposes includecolloidal dispersion systems such as for example macromoleculecomplexes, microspheres, beads, micelles and liposomes.

Generally, by means of conventional molecular biological processes it ispossible (see, e.g., Sambrook et al., supra) to introduce differentmutations into the nucleic acid molecules of the invention. As a resultthe inventive lung tumor associated polypeptides or polypeptides relatedthereto with possibly modified biological properties are synthesized.One possibility is the production of deletion mutants in which nucleicacid molecules are produced by continuous deletions from the 5′- or3′-terminal of the coding DNA sequence and that lead to the synthesis ofpolypeptides that are shortened accordingly. Another possibility is theintroduction of single-point mutation at positions where a modificationof the amino aid sequence influences, e.g., the proliferation specificproperties. By this method muteins can be produced, for example, thatpossess a modified Km-value or that are no longer subject to theregulation mechanisms that normally exist in the cell, e.g. with regardto allosteric regulation or covalent modification. Such muteins mightalso be valuable as therapeutically useful antagonists of the inventivelung tumor associated marker.

For the manipulation in prokaryotic cells by means of geneticengineering the nucleic acid molecules of the invention or parts ofthese molecules can be introduced into plasmids allowing a mutagenesisor a modification of a sequence by recombination of DNA sequences. Bymeans of conventional methods (cf. Sambrook et al., supra), bases can beexchanged and natural or synthetic sequences can be added. In order tolink the DNA fragments with each other adapters or linkers can be addedto the fragments. Furthermore, manipulations can be performed thatprovide suitable cleavage sites or that remove superfluous DNA orcleavage sites. If insertions, deletions or substitutions are possible,in vitro mutagenesis, primer repair, restriction or ligation can beperformed. As analysis method usually sequence analysis, restrictionanalysis and other biochemical or molecular biological methods are used.

The polypeptides encoded by the various variants of the nucleic acidmolecules of the invention show certain common characteristics, such asactivity in the regulation of cell proliferation and differentiation,molecular weight, immunological reactivity or conformation or physicalproperties like the electrophoretical mobility, chromatographicbehavior, sedimentation coefficients, solubility, spectroscopicproperties, stability, pH optimum, temperature optimum.

The invention furthermore relates to vectors containing the inventivelung tumor associated nucleic acid molecules. Preferably, they areplasmids, cosmids, viruses, bacteriophages and other vectors usuallyused in the field of genetic engineering. Vectors suitable for use inthe present invention include, but are not limited to the T7-based dualexpression vectors (expression in prokaryotes and in eucaryotes) forexpression in mammalian cells and baculovirus-derived vectors forexpression in insect cells. Preferably, the nucleic acid molecule of theinvention is operatively linked to the regulatory elements in therecombinant vector of the invention that guarantee the transcription andsynthesis of an mRNA in prokaryotic and/or eukaryotic cells that can betranslated. The nucleotide sequence to be transcribed can be operablylinked to a promoter like a T7, metallothionein I or polyhedrinpromoter.

In a further embodiment, the present invention relates to recombinanthost cells transiently or stably containing the nucleic acid moleculesor vectors of the invention. A host cell is understood to be an organismthat is capable to take up in vitro recombinant DNA and, if the case maybe, to synthesize the polypeptides encoded by the nucleic acid moleculesof the invention. Preferably, these cells are prokaryotic or eukaryoticcells, for example mammalian cells, bacterial cells, insect cells oryeast cells. The host cells of the invention are preferablycharacterized by the fact that the introduced nucleic acid molecule ofthe invention either is heterologous with regard to the transformedcell, i.e. that it does not naturally occur in these cells, or islocalized at a place in the genome different from that of thecorresponding naturally occurring sequence.

A further embodiment of the invention relates to a polypeptideexhibiting a biological property of the inventive lung tumor associatedmarker and being encoded by the nucleic acid molecules of the invention,as well as to methods for their production, whereby, e.g., a host cellof the invention is cultivated under conditions allowing the synthesisof the polypeptide and the polypeptide is subsequently isolated from thecultivated cells and/or the culture medium. Isolation and purificationof the recombinantly produced polypeptide may be carried out byconventional means including preparative chromatography and affinity andimmunological separations using, e.g., an antibody directed against theinventive lung tumor associated marker proteins, or, e.g., can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67; 31-40 (1988). These polypeptides, however, not onlycomprise recombinantly produced polypeptides but include isolatednaturally occurring polypeptides, synthetically produced polypeptides,or polypeptides produced by a combination of these methods. Means forpreparing such polypeptides or related polypeptides are well understoodin the art. These polypeptides are preferably in a substantiallypurified form.

The production of a polypeptide according to the present invention mayfor example be carried out in a cell free in vitro transcription and/ortranslation system. Such systems are known to those of ordinary skill inthe art. One example may comprise an intro translation system asprovided by Roche molecular Biochemicals' Rapid translation System.

Polypeptides as used in the present invention comprise at least animmunogenic portion of the inventive lung tumor associated markerproteins disclosed herein. The polypeptides may be of any length.Immunogenic portion as used above is a portion of a protein, that isrecognized by a B-cell and/or T-cell surface antigen receptor. Theimmunogenic portions comprise at least 10 amino acid residues, morepreferably at least 20 amino acid residues of the protein associatedwith a lung tumor. In a preferred embodiment of the present invention,particular domains of the proteins, such as for example transmembranedomains or N-terminal leader sequences have been deleted. Theimmunogenic portions according to the present invention react withantisera or specific antibodies in the same or nearly same intensity asthe native full length proteins.

The immunogenic portions are generally identified using the techniqueswell known in the art. Possible techniques are for example screening ofthe polypeptides for the ability to react with antigen-specificantibodies, antisera and/or T-cell lines or clones.

The polypeptides associated with lung tumors according to the presentinvention comprise also variants of the native proteins. These variantsmay differ from the native protein in one or more alterations such assubstitutions, deletions, additions and/or insertions. Theimmunoreactivity of the variants according to the present invention isnot substantially diminished compared to the native proteins. In apreferred embodiment of the invention the immunoreactivity is diminishedless than 50% in a more preferred embodiment the immunoreactivity isdiminished less than 20% compared to the native polypeptides.

In a preferred embodiment variants may be deficient in one or moreportions, such as for example N-terminal leader sequences, transmembranedomains or small N- and/or C-terminal sequences. The variants exhibit70%, more preferably at least 90% and most preferably at least 95%identity to the polypeptides disclosed according to the presentinvention.

The variants of the present invention are preferably conservativesubstitutions, so that the amino acids changed are substituted for aminoacids with similar properties. The properties concerned may includepolarity, charge, solubility, hydrophobicity, hydrophilicity and/oramphipathic nature of the amino acid residues. The variants disclosedherein may also comprise additional terminal leader sequences, linkersor sequences, which enable synthesis, purification or stability of thepolypeptides in an easier or more comfortable way.

The polypeptides according to the present invention comprise alsopolypeptides that are fusion or chimeric polypeptides comprising theamino acid sequence encoded by the nucleic acid sequence of theinventive lung tumor associated marker disclosed herein. Thepolypeptides may be fused to any suitable amino acid sequences. Thesesequences may for example comprise antigenic fragments, receptors,enzymes, toxins, chelating epitopes, etc. In a preferred embodiment ofthe present invention the amino acid sequences, that are fused to thedisclosed polypeptides are tags useful in the purification or recoveryof the polypeptides such as e.g. his-tags or myc-tags. The amino acidsequences fused together may be directly linked or may be separated byany linker or spacer sequences suitable in the particular purpose.

The polypeptides and polynucleotides according to the present inventionare isolated. This means that the molecules are removed from theiroriginal environment. Naturally occurring proteins are isolated if theyare separated from some or all of the materials, which coexist in thenatural environment. Polynucleotides are isolated for example if theyare cloned into vectors.

Furthermore, the present invention provides binding agents such asantibodies and antigen-binding fragments, that specifically bind to theproteins associated with a lung tumors disclosed herein.

The term binding agent comprises a variety of substances such asoligopeptides, antibodies, peptdiomimetic molecules comprising antigenbinding oligopeptides, nucleic acids, carbohydrates, organic compounds,etc. Antibody according to the present invention preferably relates toantibodies which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations. Monoclonal antibodies are made from an antigencontaining fragments of the polypeptides of the invention by methodswell known to those skilled in the art (see, e.g., Köhler et al., Nature256 (1975), 495). As used herein, the term “antibody” (Ab) or“monoclonal antibody” (Mab) is meant to include intact molecules as wellas antibody fragments (such as, for example, Fab and F(ab′) 2 fragments)which are capable of specifically binding to protein. Fab and f(ab′)₂fragments lack the Fc fragment of intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding thanan intact antibody. (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)).Thus, these fragments are preferred, as well as the products of a FAB orother immunoglobulin expression library. Moreover, antibodies of thepresent invention include chimerical, single chain, and humanizedantibodies.

Binding agents according to the present invention may for example beemployed for the inhibition of the activity of the inventive lung tumorassociated marker polypeptides disclosed herein. In this respect theterm “binding agents” relates to agents specifically binding to thepolypeptides transcribed from the novel lung tumor associated nucleicacids and thus inhibiting the activity of said polypeptide. Such bindingagents may for example comprise nucleic acids (DNA, RNA, PNA etc.),polypeptides (antibodies, receptors, antigenic fragments,oligopeptides), carbohydrates, lipids, organic or inorganic compounds(metal-ions, sulfur compounds, boranes, silicates, reducing agents,oxidizing agents). The binding agents may preferably interact with thepolypeptide by binding to epitopes, that are essential for thebiological activity. The interaction may be reversible or irreversibly.The binding may be non-covalent or even covalent binding to thepolypeptide. Furthermore the binding agents may introduce alterations tothe polypeptide, that alter or diminish the biological activity of theinventive polypeptide.

For certain purposes, e.g. diagnostic methods, the antibody or bindingagent of the present invention may be detectably labelled, for example,with a radioisotope, a bioluminescent compound, a chemiluminescentcompound, a fluorescent compound, a metal chelate, or an enzyme. Thenucleic acid of the present invention may be detectably labelled, forexample, with a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate,biotin, digoxygenin, or an enzyme. Furthermore any method suitable forthe detection of the intermolecular interaction may be employed.

The antibody or antigen-binding agent is said to react specifically, ifit reacts at a detectable level with a protein disclosed herein, anddoes not significantly react with other proteins. The antibodiesaccording to the present invention may be monoclonal or polyclonalantibodies. Other molecules capable of binding specifically may be forexample antigen-binding fragments of antibodies such as Fab fragments,RNA molecules or polypeptides. According to the present inventionbinding agents may be used isolated or in combination. By means ofcombination it is possible to achieve a higher degree of sensitivity.

The antibodies useful for the methods according to the present inventionmay comprise further binding sites for either therapeutic agents orother polypeptides or may be coupled to said therapeutic agents orpolypeptides. Therapeutic agents may comprise drugs, toxins,radio-nuclides and derivatives thereof. The agents may be coupled to thebinding agents either directly or indirectly for example by a linker orcarrier group. The linker group may for example function in order toenable the coupling reaction between binding agent and therapeutic orother agent or the linker may act as a spacer between the distinct partsof the fusion molecule. The linker may also be cleavable under certaincircumstances, so as to release the bound agent under said conditions.The therapeutic agents may be covalently coupled to carrier groupsdirectly or via a linker group. The agent may also be non-covalentlycoupled to the carrier. Carriers that can be used according to thepresent invention are for example albumins, polypeptides,polysaccharides or liposomes.

The antibody used according to the present invention may be coupled toone or more agents. The multiple agents coupled to one antibody may beall of the same species or may be several different agents bound to oneantibody.

The invention also relates to a transgenic non-human animal such astransgenic mouse, rats, hamsters, dogs, monkeys, rabbits, pigs, C.elegans and fish such as torpedo fish comprising a nucleic acid moleculeor vector of the invention, preferably wherein said nucleic acidmolecule or vector may be stably integrated into the genome of saidnon-human animal, preferably such that the presence of said nucleic acidmolecule or vector leads to the expression of the inventive lung tumorassociated marker polypeptide (or related polypeptide) of the invention,or may otherwise be transiently expressed within the non-human animal.Said animal may have one or several copies of the same or differentnucleic acid molecules encoding one or several forms of the inventivelung tumor associated marker polypeptide or mutant forms thereof. Thisanimal has numerous utilities, including as a research model for theregulation of cell proliferation and differentiation and therefore,presents a novel and valuable animal in the development of therapies,treatment, etc. for diseases caused by deficiency or failure of theinventive lung tumor associated marker protein involved in thedevelopment of cell proliferative disorders, e.g., lung tumors.Accordingly, in this instance, the non-human mammal is preferably alaboratory animal such as a mouse or rat.

Preferably, the transgenic non-human animal of the invention furthercomprises at least one inactivated wild type allele of the correspondinggene encoding the inventive lung tumor associated polypeptide. Thisembodiment allows for example the study of the interaction of variousmutant forms of the inventive lung tumor associated marker polypeptideson the onset of the clinical symptoms of disease associated with theregulation of cell proliferation and differentiation. All theapplications that have been herein before discussed with regard to atransgenic animal also apply to animals carrying two, three or moretransgenes. It might be also desirable to inactivate the inventive lungtumor associated marker protein expression or function at a certainstage of development and/or life of the transgenic animal. This can beachieved by using, for example, tissue specific, developmental and/orcell regulated and/or inducible promoters which drive the expression of,e.g., an antisense or ribozyme directed against the RNA transcriptencoding the inventive lung tumor associated marker encoding mRNA; seealso supra. A suitable inducible system is for exampletetracycline-regulated gene expression as described, e.g., by Gossen andBujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen etal. (Trends Biotech. 12 (1994), 58-62). Similar, the expression of themutant inventive lung tumor associated protein may be controlled by suchregulatory elements.

Furthermore, the invention also relates to a transgenic mammalian cellwhich contains (preferably stably integrated into its genome ortransiently introduced) a nucleic acid molecule according to theinvention or part thereof, wherein the transcription and/or expressionof the nucleic acid molecule or part thereof leads to reduction of thesynthesis of an inventive lung tumor associated marker protein. In apreferred embodiment, the reduction is achieved by an anti-sense, sense,ribozyme, co-suppression and/or dominant mutant effect. “Antisense” and“antisense nucleotides” means DNA or RNA constructs which block theexpression of the naturally occurring gene product. In another preferredembodiment the native nucleic acid sequence coding for the inventivelung tumor associated marker polypeptide may be altered or substitutedby a variant of said nucleic acid sequence, e.g. by means ofrecombination, thus rendering the inventive lung tumor associated markergene non functional. Thus an organism lacking the inventive lung tumorassociated marker polypeptide activity may be produced according toknock out experiments.

The provision of the nucleic acid molecule according to the inventionopens up the possibility to produce transgenic non-human animals with areduced level of the inventive lung tumor associated marker protein asdescribed above and, thus, with a defect in the regulation of cellproliferation and differentiation. Techniques how to achieve this arewell known to the person skilled in the art. These include, for example,the expression of antisense-RNA, ribozymes, of molecules which combineantisense and ribozyme functions and/or of molecules which provide for aco-suppression effect. When using the antisense approach for reductionof the amount of the inventive lung tumor associated marker proteins incells, the nucleic acid molecule encoding the antisense-RNA ispreferably of homologous origin with respect to the animal species usedfor transformation. However, it is also possible to use nucleic acidmolecules which display a high degree of homology to endogenouslyoccurring nucleic acid molecules encoding an inventive lung tumorassociated marker protein. In this case the homology is preferablyhigher than 80%, particularly higher than 90% and still more preferablyhigher than 95%. The reduction of the synthesis of a polypeptideaccording to the invention in the transgenic mammalian cells can resultin an alteration in, e.g., degradation of endogenous proteins. Intransgenic animals comprising such cells this can lead to variousphysiological, developmental and/or morphological changes.

Thus, the present invention also relates to transgenic non-human animalscomprising the above-described transgenic cells. These may show, forexample, a deficiency in regulation of cell proliferation and/ordifferentiation compared to wild type animals due to the stable ortransient presence of a foreign DNA resulting in at least one of thefollowing features:

-   -   (a) disruption of (an) endogenous gene(s) encoding the inventive        lung tumor associated marker;    -   (b) expression of at least one antisense RNA and/or ribozyme        against a transcript comprising a nucleic acid molecule of the        invention;    -   (c) expression of a sense and/or non-translatable mRNA of the        nucleic acid molecule of the invention;    -   (d) expression of an antibody of the invention;    -   (e) incorporation of a functional or non-functional copy of the        regulatory sequence of the invention; or    -   (f) incorporation of a recombinant DNA molecule or vector of the        invention.

Methods for the production of a transgenic non-human animal of thepresent invention, preferably transgenic mouse, are well known to theperson skilled in the art. Such methods, e.g., comprise the introductionof a nucleic acid molecule or vector of the invention into a germ cell,an embryonic cell, stem cell or an egg or a cell derived therefrom. Thenon-human animal can be used in accordance with a screening method ofthe invention described herein and may be a non-transgenic healthyanimal, or may have a disorder, preferably a disorder caused by at leastone mutation in the inventive lung tumor associated marker protein. Suchtransgenic animals are well suited for, e.g., pharmacological studies ofdrugs in connection with mutant forms of the above described inventivelung tumor associated marker polypeptide. Production of transgenicembryos and screening of those can be performed, e.g., as described byA. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), OxfordUniversity Press. The DNA of the embryonal membranes of embryos can beanalyzed using, e.g., Southern blots with an appropriate probe,amplification techniques based on nucleic acids (e.g. PCR) etc.; seesupra.

Another aspect of the present invention is a pharmaceutical compositionfor use in the treatment of disorders associated with abnormal cellproliferation. The polypeptides, polynucleotides and binding agents(esp. antibodies) according to the present invention may be incorporatedinto pharmaceutical or immunogenic compositions.

The pharmaceutical compositions may be administered by any suitable wayknown to those of skill in the art. The administration may for examplecomprise injection, such as e.g., intracutaneous, intramuscular,intravenous or subcutaneous injection, intranasal administration forexample by aspiration or oral administration. A suitable dosage toensure the pharmaceutical benefit of the treatment should be chosenaccording the parameters, such as age, sex, body weight etc. of thepatient, known to those of skill in the art.

The pharmaceutical compositions comprise said compounds and aphysiologically acceptable carrier. The type of carrier to be employedin the pharmaceutical compositions of this invention, will varydepending on the mode of administration. For parenteral administration,such as subcutaneous injection, the carrier preferably comprises water,saline, alcohol, a lipid, a wax and/or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and/or magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic glycolide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

A pharmaceutical composition or vaccine may for example contain DNA,that codes for one or more polypeptides according to the presentinvention. The DNA may be administered in a way that allows thepolypeptides to be generated in situ. Suitable expression systems areknown to those skilled in the art. In another embodiment of theinvention the nucleic acids may be for example anti-sense constructs.Pharmaceutical compositions may also comprise nucleic acid moleculesexpressible in a mammalian or human host system comprising a viral orother expression system for example an adenoviral vector system.

The nucleic acid may also be administered as a naked nucleic acid. Inthis case appropriate physical delivery systems, which enhance theuptake of nucleic acid may be employed, such as coating the nucleic acidonto biodegradable beads, which are efficiently transported into thecells. Administration of naked nucleic acids may for example be usefulfor the purpose of transient expression within a host or host cell.

Alternatively the pharmaceutical compositions may comprise one or morepolypeptides. The polypeptides incorporated into pharmaceuticalcompositions may be the inventive lung tumor associated polypeptide.Optionally the polypeptide may be administered in combination with oneor more other known polypeptides such as for example enzymes,antibodies, regulatory factors, such as cyclins, cyclin-dependentkinases or CKIs, or toxins.

Polypeptides of the present invention or fragments thereof, thatcomprise an immunogenic portion of an inventive lung tumor associatedprotein, may be used in immunogenic compositions, wherein thepolypeptide e.g. stimulates the patient's own immune response to tumorcells. A patient may be afflicted with disease, or may be free ofdetectable disease. Accordingly, the compounds disclosed herein may beused to treat cancer or to inhibit the development of cancer. Thecompounds may be administered either prior to or following aconventional treatment of tumors such as surgical removal of primarytumors, treatment by administration of radiotherapy, conventionalchemotherapeutic methods or any other mode of treatment of therespective cancer or its precursors.

Immunogenic compositions such as vaccines may comprise one or morepolypeptides and a non-specific immune-response enhancer, wherein thenon-specific immune response enhancer is capable of eliciting orenhancing an immune response to an exogenous antigen. Any suitableimmune-response enhancer may be employed in the vaccines of thisinvention. For example, an adjuvant may be included. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminium hydroxide or mineral oil, and anon-specific stimulator of immune response, such as lipid A, Bordetellapertussis or Mycobacterium tuberculosis. Such adjuvants are commerciallyavailable as, for example, Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.) and Merck Adjuvant 65(Merck and Company, Inc., Rahway, N.J.).

Pharmaceutical compositions and vaccines may also contain other epitopesof tumor antigens, either incorporated into a fusion protein asdescribed above (i.e., a single polypeptide that contains multipleepitopes) or present within a separate polypeptide.

Disorders characterized by abnormal cell proliferation, as used in thecontext of the present invention, may comprise for example neoplasmssuch as benign and malignant tumors, carcinomas, sarcomas, leukemias,lymphomas or dysplasias. Tumors may comprise tumors of the head and theneck, tumors of the respiratory tract, tumors of the gastrointestinaltract, tumors of the urinary system, tumors of the reproductive system,tumors of the endocrine system, tumors of the central and peripheralnervous system, tumors of the skin and its appendages, tumors of thesoft tissues and bones, tumors of the lymphopoietic and hematopoieticsystem, breast cancer, colorectal cancer, anogenital cancer etc.

In one preferred embodiment of the invention, the disorders are lungtumors. Lung tumors according to the present invention compriseconditions of the respiratory tract characterized by abnormal growthproperties of cells or tissues compared to the growth properties ofnormal control cells or tissues. The growth of the cells or tissues maybe for example abnormally accelerated or may be regulated abnormally.Abnormal regulation as used above may comprise any form of presence orabsence of non-wild type responses of the cells or tissues to naturallyoccurring growth regulating influences. The abnormalities in growth ofthe cells or tissues may be for example neoplastic or hyperplastic. Inone preferred embodiment of the invention the lung tumors are cancers orprecancerous conditions of the respiratory tract.

A sample according to the method of the present invention is any sample,that may contain cells, tissues or body liquids. Furthermore any samplepotentially containing the marker molecules to be detected may be asample according to the present invention. Such samples are e.g. blood,plasma, serum, swabs, washes, sputum, cell- and tissue-samples orbiopsies.

Biopsies as used in the context of the present invention may comprisee.g. resection samples of tumors, tissue samples prepared by endoscopicmeans or needle biopsies. Furthermore any sample potentially containingthe marker molecules to be detected may be a sample according to thepresent invention.

The method for detection of the level of the polynucleotides orpolypeptides according to the present invention is any method, which issuited to detect very small amounts of specific molecules in samples.The detection reaction according to the present invention may be forexample a detection either on the level of nucleic acids or on the levelof polypeptides. The detection may either be a detection of the level ofpolypeptides or nucleic acids in cells in total or in cell lysates or adetection of the level of polypeptides or nucleic acids in distinctsubcellular regions. The methods for determining the subcellulardistribution of compounds are known to those of skill in the art. In oneembodiment of the invention the detection of the marker molecules maycomprise the detection of particular splicing variants. In anotherembodiment of the present invention the detection method may comprisethe detection of methylation of nucleic acid molecules in samples.

Applicable formats for the detection reaction according to the presentinvention may be blotting techniques, such as Western-Blot,Southern-blot, Northern-blot. The blotting techniques are known to thoseof ordinary skill in the art and may be performed for example aselectro-blots, semidry-blots, vacuum-blots or dot-blots. Furthermoreimmunological methods for detection of molecules may be applied, such asfor example immunoprecipitation or immunological assays, such as ELISA,RIA, lateral flow assays etc.

Methods for detection of methylation of nucleic acids are known to thoseof skill in the art and may comprise for example methods employingchemical pre-treatment of nucleic acids with e.g. sodium bisulphite,permanganate or hydrazine, and subsequent detection of the modificationby means of specific restriction endonucleases or by means of specificprobes e.g. in the course of an amplification reaction. The detection ofmethylation may furthermore be performed using methylation specificrestriction endonucleases.

In one preferred embodiment of the invention, the detection of the levelof marker molecules is carried out by detection of the level of nucleicacids coding for the marker molecules or fragments thereof present inthe sample. The means for detection of nucleic acid molecules are knownto those skilled in the art. The procedure for the detection of nucleicacids can for example be carried out by a binding reaction of themolecule to be detected to complementary nucleic acid probes, proteinswith binding specificity for the nucleic acids or any other entitiesspecifically recognizing and binding to said nucleic acids. This methodcan be performed as well in vitro as directly in-situ for example in thecourse of a detecting staining reaction. Another way of detecting themarker molecules in a sample on the level of nucleic acids performed inthe method according to the present invention is an amplificationreaction of nucleic acids, which can be carried out in a quantitativemanner such as for example PCR, LCR or NASBA.

In another preferred embodiment of the invention the detection of thelevel of marker molecules is carried out by determining the level ofexpression of a protein. The determination of the marker molecules onthe protein level may for example be carried out in a reactioncomprising a binding agent specific for the detection of the markermolecules. These binding agents may comprise for example antibodies andantigen-binding fragments, bifunctional hybrid antibodies,peptidomimetics containing minimal antigen-binding epitopes etc. Thebinding agents may be used in many different detection techniques forexample in western-blot, ELISA, lateral flow assay, latex-agglutination,immunochromatographic strips or immuno-precipitation. Generally bindingagent based detection may be carried out as well in vitro as directly insitu for example in the course of an immuno-cytochemical stainingreaction. Any other method suitable for determining the amount ofparticular polypeptides in solutions of biological samples, such asbiochemical, chemical, physical or physico-chemical methods, can be usedaccording to the present invention.

In one preferred embodiment of the invention, the level of markers issignificantly elevated compared to a non tumorous test sample. In thiscase the marker is overexpressed in the sample. In another preferredembodiment of the present invention the level of the marker is loweredcompared to a non tumorous test sample. In a third embodiment there isno detectable expression of the marker at all in the test sample unlikein a control sample. In yet another embodiment there is detectable levelof non wild-type marker molecules. Non wild-Type marker molecules maycomprise any marker molecules that deviate in sequence or structure fromthe structure or sequence, that is functional in wild type tissue notaffected by a cell proliferative disease. Wild type sequences orstructures are the sequences or structures predominantly present innormal cells or tissues. In one preferred embodiment of the inventionthe level of particular splicing variants of the marker gene is alteredin the test samples compared to the wild type tissue. This may lead toaltered levels of splicing variants, new splicing variants,neo-peptides, altered ratios of different splicing variants of genes.

The detection of the level of molecular markers according to the presentinvention may be the detection of the level of single marker moleculesin separated reaction mixtures as well as the detection of a combinationof markers simultaneously. The combination may comprise the molecularmarkers disclosed herein and additionally further marker moleculesuseful for the detection of tumors.

The detection may be carried out in solution or using reagents fixed toa solid phase. The detection of one or more molecular markers may beperformed in a single reaction mixture or in two or separate reactionmixtures. The detection reactions for several marker molecules may forexample be performed simultaneously in multi-well reaction vessels. Themarkers characteristic for the lung tumors disclosed herein may bedetected using reagents that specifically recognise these molecules.Simultaneously one or more further markers may be detected usingreagents, that specifically recognize them. The detection reaction foreach single marker may comprise one or more reactions with detectingagents either recognizing the initial marker molecules or preferablyrecognizing molecules used to recognize other molecules. Such reactionmay e.g. comprise the use of primary and secondary and furtherantibodies. The detection reaction further may comprise a reporterreaction indicating the level of the inventive polypeptides associatedwith lung tumors. The reporter reaction may be for example a reactionproducing a coloured compound, a bioluminescence reaction, afluorescence reaction, generally a radiation emitting reaction etc.

In one preferred embodiment, the detection of tissues expressing markergene products is carried out in form of molecular imaging procedures.The respective procedures are known to those of ordinary skill in theart. Imaging methods for use in the context of the present invention mayfor example comprise MRI, SPECT, PET and other methods suitable for invivo imaging.

In one embodiment the method may be based on the enzymatic conversion ofinert or labelled compounds to molecules detectable in the course ofmolecular imaging methods by the marker molecules. In another embodimentthe molecular imaging method may be based on the use of compoundscarrying a suitable label for in vivo molecular imaging, such as radioisotopes, metal ions etc., specifically binding to marker molecules invivo.

In a preferred embodiment of the invention, these compounds arenon-toxic compounds and may be eliminated from the circulation oforganisms, such as humans, in a time span, that allows for performingthe detection of label accumulated in tumor tissue overexpressing therespective marker gene. In another preferred embodiment of the inventioncompounds are used for molecular imaging, for which clearance from thecirculation is not relevant for performing the molecular imagingreaction. This may be for example due to low background produced by thecirculating molecules etc. The compounds for use in molecular imagingmethods are administered in pharmaceutical acceptable form incompositions that may additionally comprise any other suitablesubstances, such as e.g. other diagnostically useful substances,therapeutically useful substances, carrier substances or the like.

The marker molecules disclosed according to the present invention may beused for diagnosis, monitoring of the disease course and prognosis incell proliferative disorders such as e.g. lung tumors.

Diagnosis of disorders associated with the expression of the inventivegene as used herein may for example comprise the detection of cells ortissues affected by abnormal growth. In one preferred embodimentdiagnosis means the primary detection of a disease in an organism orsample. According to the present invention the method for diagnosis ofdisorders such as tumors may be applied in routine screening tests forpreventive aspects in order to detect said disease at an early stage ofthe onset of the disorder. In another preferred embodiment thediagnostic method may be used to determine the minimal residual diseaseof a tumor after primary therapy. In this respect the method of theinvention may be applied to determine cells in body samples displayingabnormal expression of marker molecules according to the presentinvention, characteristic for lung tumors. Thus a spread of affectedcells may be detected in body liquids.

In one embodiment of the invention, the methods disclosed herein may beused for the detection and identification of metastases. The method maybe applied either for detection of metastases in body tissues or organsby the detection methods described herein, or the metastases may bediagnoses with respect to prognosis and prediction of disease course.

Monitoring of the disease course may comprise determining the levels ofmarker molecules at different time points, comparing the levels at thedifferent time points and assessing a diagnosis about the progression ofthe disease over the covered period of time. Thus monitoring may enablefor assessment of prognosis and/or for design of an adequate therapy fora particular patient.

Prognosis of the disease course of a cell proliferative disorders suchas e.g. lung tumors according to the present invention may comprisedetermining the level of expression of one or more marker molecules,comparing the levels with data from subsequent studies in a database andprognosticating the disease course from said comparison. In a preferredembodiment the method may comprise the detection of the levels of a setof marker molecules, the distinct levels of which may characterizedistinct stages in the course of the disease. In a further embodiment ofthe invention the combination of the levels of a combination of markersmay be an indicator for the prognosis of the further disease course andmay build the basis for design of an adequate therapy.

The present invention further provides kits for use in e.g. research ordiagnostic methods. Such kits may contain two or more components forperforming a scientific or diagnostic assay. Components may becompounds, reagents, containers and/or equipment. One component may bean antibody or fragment thereof that specifically binds to a polypeptideassociated with lung tumors. Additionally the kit may contain reagents,buffers or others known in the art as necessary for performing thediagnostic assay. Alternatively the research kit or diagnostic kit maycontain nucleotide probes or primers for the detection of DNA or RNA.Such a kit should contain appropriate additional reagents and buffersknown in the art.

A kit according to present invention comprises:

-   -   a) reagents for the detection of the molecular marker molecules,    -   b) the reagents and buffers commonly used for carrying out the        detection reaction, such as buffers, detection-markers, carrier        substances and others, and    -   c) a marker sample for carrying out a positive control reaction.

The reagent for the detection of the marker includes any agent capableof binding to the marker molecule. Such reagents may include proteins,polypeptides, nucleic acids, glycoproteins, proteoglycans,polysaccharides or lipids.

The sample for carrying out a positive control may comprise for examplenucleic acids in applicable form, such as solution or salt, peptides inapplicable form, tissue section samples or positive cells expressing themolecules associated with lung tumors.

In a preferred embodiment of the invention, the detection of the markermolecules is carried out on the level of polypeptides. In thisembodiment, the binding agents may be for example antibodies specificfor the marker molecules or fragments thereof.

In another embodiment of the test kit, the detection of the markermolecule is carried out on the nucleic acid level. In this embodiment ofthe invention the reagents for the detection may be for example nucleicacid probes or primers complementary to said marker molecule nucleicacids.

Another aspect of the present invention is to provide a method fortherapy and/or vaccination. According to the present invention a therapyof cell proliferative disorders can be carried out using the inventivelung tumor associated polypeptides and/or polynucleotides. The therapymay be for example immunotherapy or somatic gene therapy.

The inventive lung tumor associated polypeptides and/or polynucleotidesmay according to the present invention be used for vaccination againstcell proliferative disorders. Vaccination according to the presentinvention may comprise administering an immunogenic compound to anindividual for the purpose of stimulating an immune response directedagainst said immunogenic compound and thus immunizing said individualagainst said immunogenic compound. Stimulating an immune response maycomprise inducing the production of antibodies against said compound aswell as stimulating cytotoxic T-cells. For the purpose of vaccinationthe polypeptides, nucleic acids and binding agents according to thepresent invention may be administered in a physiological acceptableform. The composition to be administered to individuals may comprise oneor more antigenic components, physiologically acceptable carriersubstances or buffer solutions, immunostimulants and/or adjuvants.Adjuvants may comprise for example Freund's incomplete adjuvant orFreund's complete adjuvant or other adjuvants known to those of skill inthe art.

The composition may be administered in any applicable way such as e.g.intravenous, subcutaneous, intramuscular etc. The dosage of thecomposition depends on the particular case and purpose of thevaccination. It has to be adapted to parameters by the individualtreated such as age, weight, sex etc. Furthermore the type of the immuneresponse to be elicited has to be taken into account. In general it maybe preferable if an individual receives 100 μg-1 g of a polypeptideaccording to the present invention or 10⁶-10¹² MOI of a recombinantnucleic acid, containing a nucleic acid according to the presentinvention in a form that may be expressed in situ.

Individuals for the purpose of vaccination may be any organismscontaining the inventive lung tumor associated polypeptides and/orpolynucleotides and being able to get affected by cell proliferativedisorders.

Vaccination of individuals may be favourable e.g. in the case ofaltered, non wild-type sequences or structure of marker moleculesassociated with cell proliferative disorders.

Polypeptides disclosed herein may also be employed in adoptiveimmunotherapy for the treatment of cancer. Adoptive immunotherapy may bebroadly classified into either active or passive immunotherapy. Inactive immunotherapy, treatment relies on the in vivo stimulation of theendogenous host immune system to react against tumors with theadministration of immune response-modifying agents (for example, tumorvaccines, bacterial adjuvants, and/or cytokines).

In passive immunotherapy, treatment involves the delivery of biologicreagents with established tumor-immune reactivity (such as effectorcells or antibodies) that can directly or indirectly mediate antitumoreffects and does not necessarily depend on an intact host immune system.Examples of effector cells include T lymphocytes (for example, CD8+cytotoxic T-lymphocyte, CD4+ T-helper, tumor-infiltrating lymphocytes),killer cells (such as Natural Killer cells, lymphokine-activated killercells), B cells, or antigen presenting cells (such as dendritic cellsand macrophages) expressing the disclosed antigens. The polypeptidesdisclosed herein may also be used to generate antibodies oranti-idiotypic antibodies (as in U.S. Pat. No. 4,918,164), for passiveimmunotherapy.

The predominant method of procuring adequate numbers of T-cells foradoptive immunotherapy is to grow immune T-cells in vitro. Cultureconditions for expanding single antigen-specific T-cells to severalbillion in number with retention of antigen recognition in vivo are wellknown in the art. These in vitro culture conditions typically utilizeintermittent stimulation with antigen, often in the presence ofcytokines, such as IL-2, and non-dividing feeder cells. As noted above,the immunoreactive polypeptides described herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate sufficientnumber of cells for immunotherapy. In particular, antigen-presentingcells, such as dendritic, macrophage or B-cells, may be pulsed withimmunoreactive polypeptides or transfected with a nucleic acidsequence(s), using standard techniques well known in the art. Forexample, antigen presenting cells may be transfected with a nucleic acidsequence, wherein said sequence contains a promoter region appropriatefor increasing expression, and can be expressed as part of a recombinantvirus or other expression system. For cultured T-cells to be effectivein therapy, the cultured T-cells must be able to grow and distributewidely and to survive long term in vivo. Studies have demonstrated thatcultured T-cells can be induced to grow in vivo and to survive long termin substantial numbers by repeated stimulation with antigen supplementedwith IL-2 (see, for example, Cheever, M., et al, “Therapy With CulturedT Cells: Principles Revisited,” Immunological Reviews, 157:177, 1997).

The polypeptides disclosed herein may also be employed to generateand/or isolate tumor-reactive T-cells, which can then be administered tothe patient. In one technique, antigen-specific T-cell lines may begenerated by in vivo immunization with short peptides corresponding toimmunogenic portions of the disclosed polypeptides. The resultingantigen specific CD8+ CTL clones may be isolated from the patient,expanded using standard tissue culture techniques, and returned to thepatient.

Alternatively, peptides corresponding to immunogenic portions of thepolypeptides of the invention may be employed to generate tumor reactiveT-cell subsets by selective in vitro stimulation and expansion ofautologous T-cells to provide antigen-specific T-cells which may besubsequently transferred to the patient as described, for example, byChang et al. (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996). Cells ofthe immune system, such as T-cells, may be isolated from the peripheralblood of a patient, using a commercially available cell separationsystem, such as CellPro Incorporated's (Bothell, Wash.) CEPRATE™ system(see U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO91/16116 and WO 92/07243). The separated cells are stimulated with oneor more of the immunoreactive polypeptides contained within a deliveryvehicle, such as a microsphere, to provide antigen-specific T-cells. Thepopulation of tumor antigen-specific T-cells is then expanded usingstandard techniques and the cells are administered back to the patient.

In another embodiment, T-cell and/or antibody receptors specific for thepolypeptides can be cloned, expanded, and transferred into other vectorsor effector cells for use in adoptive immunotherapy.

In a further embodiment, syngeneic or autologous dendritic cells may bepulsed with peptides corresponding to at least an immunogenic portion ofa polypeptide disclosed herein. The resulting antigen-specific dendriticcells may either be transferred into a patient, or employed to stimulateT-cells to provide antigen-specific T-cells, which may, in turn, beadministered to a patient. The use of peptide-pulsed dendritic cells togenerate antigen-specific T-cells and the subsequent use of suchantigen-specific T-cells to eradicate tumors in a murine model has beendemonstrated by Cheever et al, Immunological Reviews, 157:177, 1997.

Additionally, vectors expressing the disclosed nucleic acids may beintroduced into stem cells taken from the patient and clonallypropagated in vitro for autologous transplant back into the samepatient.

Monoclonal antibodies of the present invention may also be used astherapeutic compounds in order to diminish or eliminate tumors. Theantibodies may be used on their own (for instance, to inhibitmetastases) or coupled to one or more therapeutic agents. Suitableagents in this regard include radio nuclides, differentiation inducers,drugs, toxins, and derivatives thereof. Preferred radio nuclides include90Y, 123I, 125I, 131I, 186Re, 188Re, 211At, and 212Bi. Preferred drugsinclude methotrexate, and pyrimidine and purine analogs. Preferreddifferentiation inducers include phorbol esters and butyric acid.Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin,gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviralprotein.

In one embodiment of the invention, the therapy of disorderscharacterized by abnormal cell proliferation may comprise theadministration of antisense construct or ribozymes. The methods foradministration of ribozymes or antisense constructs are known to thoseof skill in the art. The administration may take place as administrationof naked nucleic acids or as administration of nucleic acids that aresuited for expression of the relevant active products in situ.

In another embodiment of the invention the treatment of disorders maycomprise the administration of binding agents directed against theinventive lung tumor associated molecules. These binding agents may forexample be coupled to other compounds such as toxins, enzymes,radio-isotopes etc.

In another embodiment of the invention, therapy of disorders associatedwith abnormal expression of the presented inventive lung tumorassociated molecules may comprise the administration of antagonists oragonists of the inventive lung tumor associated molecules, of bindingpartners of the inventive lung tumor associated polypeptides ofinhibitors or enhancer of the expression of the inventive lung tumorassociated polypeptides or of drugs identifiable by assays involving themeasurement of the activity of the inventive lung tumor associatedpolypeptides. The methods for identifying these substances are known tothose of skill in the art.

An example for a method for identifying a binding partner of aninventive lung tumor associated polypeptides (or related polypeptide)and/or polynucleotide may comprise:

-   -   (a) contacting the inventive lung tumor associated polypeptide        of the invention with a compound to be screened; and    -   (b) determining whether the compound affects an activity of the        polypeptide.

The inventive lung tumor associated polypeptides may be used to screenfor proteins or other compounds that bind to the inventive lung tumorassociated polypeptides or for proteins or other compounds to which theinventive lung tumor associated polypeptide binds. The binding of theinventive lung tumor associated polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the inventive lung tumor associated polypeptide or the moleculebound. Examples of such molecules include antibodies, oligonucleotides,proteins (e.g., receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of theinventive lung tumor associated polypeptide, e.g., a fragment of theligand, or a natural substrate, a ligand, a structural or functionalmimetic; see, e.g., Coligan, Current Protocols in Immunology 1(2)(1991); Chapter 5. Similarly, the molecule can be closely related to thenatural receptor to which the inventive lung tumor associatedpolypeptide might bind, or at least, a fragment of the receptor capableof being bound by the inventive lung tumor associated polypeptide (e.g.,active site). In either case, the molecule can be rationally designedusing known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the inventive lung tumor associatedpolypeptide, either as a secreted protein or on the cell membrane.Preferred cells include cells from mammals, yeast, Drosophila, or E.coli. Cells expressing the inventive lung tumor associated polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofthe inventive lung tumor associated polypeptide.

The assay may simply test binding of a candidate compound to theinventive lung tumor associated polypeptide, wherein binding is detectedby a label, or in an assay involving competition with a labelledcompetitor. Further, the assay may test whether the candidate compoundresults in a signal generated by binding to the inventive lung tumorassociated polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining the inventive lung tumor associated polypeptide, measuringthe inventive lung tumor associated polypeptide/molecule activity orbinding, and comparing the inventive lung tumor associatedpolypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure the inventive lung tumorassociated polypeptide level or activity in a sample (e.g., biologicalsample) using a monoclonal or polyclonal antibody. The antibody canmeasure the inventive lung tumor associated polypeptide level oractivity by either binding, directly or indirectly, to the inventivelung tumor associated polypeptide or by competing with the inventivelung tumor associated polypeptide for a substrate. All of these aboveassays can be used as diagnostic or prognostic markers. The moleculesdiscovered using these assays can be used to treat disease or to bringabout a particular result in a patient (e.g., elimination of anepithelial tumor or stop of progression of tumor growth) by activatingor inhibiting the inventive lung tumor associated molecule. Moreover,the assays can discover agents which may inhibit or enhance theproduction of the inventive lung tumor associated polypeptide fromsuitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compoundswhich bind to the inventive lung tumor associated polypeptide comprisingthe steps of: (a) incubating a candidate binding compound with apolypeptide of the invention (the inventive lung tumor associatedpolypeptide); and (b) determining if binding has occurred.

Moreover, the invention includes a method of identifyingactivators/agonists or inhibitors/antagonists of a the inventive lungtumor associated polypeptide comprising the steps of: (a) incubating acandidate compound with a polypeptide of the invention; b) assaying abiological activity, and (c) determining if a biological activity of thepolypeptide of the invention has been altered.

In a further embodiment, the present invention relates to method ofidentifying and obtaining a drug candidate for therapy of a disordercharacterized by abnormal cell proliferation comprising the steps of

-   -   (a) contacting a lung tumor associated polypeptide of the        present invention or a cell expressing said polypeptide in the        presence of components capable of providing a detectable signal        in response to altered regulation of cell proliferation or to        altered cell differentiation, with said drug candidate to be        screened under conditions to allow protein degradation, and    -   (b) detecting presence or absence of a signal or increase of the        signal generated from protein degradation, wherein the presence        or increase of the signal is indicative for a putative drug.

Experiments using animals or isolated cells or cell lines may be used toexamine the proliferative behavior of cells or tissues in dependence onthe inventive lung tumor associated polypeptide action. The sameprocedures may be employed for the study of cell differentiation.

The drug candidate may be a single compound or a plurality of compounds.The term “plurality of compounds” in a method of the invention is to beunderstood as a plurality of substances which may or may not beidentical.

Said compound or plurality of compounds may be chemically synthesized ormicrobiologically produced and/or comprised in, for example, samples,e.g., cell extracts from, e.g., plants, animals or microorganisms.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating the inventive lungtumor associated polypeptides. The reaction mixture may be a cell freeextract or may comprise a cell or tissue culture. Suitable set ups forthe method of the invention are known to the person skilled in the artand are, for example, generally described in Alberts et al., MolecularBiology of the Cell, third edition (1994) and in the appended examples.The plurality of compounds may be, e.g., added to the reaction mixture,culture medium, injected into a cell or otherwise applied to thetransgenic animal. The cell or tissue that may be employed in the methodof the invention preferably is a host cell, mammalian cell or non-humantransgenic animal of the invention described in the embodimentshereinbefore.

If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating the inventive lungtumor associated polypeptide, or one can further subdivide the originalsample, for example, if it consists of a plurality of differentcompounds, so as to reduce the number of different substances per sampleand repeat the method with the subdivisions of the original sample.Depending on the complexity of the samples, the steps described abovecan be performed several times, preferably until the sample identifiedaccording to the method of the invention only comprises a limited numberof or only one substance(s). Preferably said sample comprises substancesof similar chemical and/or physical properties, and most preferably saidsubstances are identical.

Several methods are known to the person skilled in the art for producingand screening large libraries to identify compounds having specificaffinity for a target. These methods include the phage-display method inwhich randomized peptides are displayed from phage and screened byaffinity chromatography to an immobilized receptor; see, e.g., WO91/17271, WO 92/01047, U.S. Pat. No. 5,223,409. In another approach,combinatorial libraries of polymers immobilized on a chip aresynthesized using photolithography; see, e.g., U.S. Pat. No. 5,143,854,WO 90/15070 and WO 92/10092. The immobilized polymers are contacted witha labelled receptor and scanned for label to identify polymers bindingto the receptor. The synthesis and screening of peptide libraries oncontinuous cellulose membrane supports that can be used for identifyingbinding ligands of the polypeptide of the invention and thus possibleinhibitors and activators is described, for example, in Kramer, MethodsMol. Biol. 87 (1998), 25-39. This method can also be used, for example,for determining the binding sites and the recognition motifs in thepolypeptide of the invention. In like manner, the substrate specificityof the DnaK chaperon was determined and the contact sites between humaninterleukin-6 and its receptor; see Rudiger, EMBO J. 16 (1997),1501-1507 and Weiergraber, FEBS Lett. 379 (1996), 122-126, respectively.Furthermore, the above-mentioned methods can be used for theconstruction of binding supertopes derived from the polypeptide of theinvention. A similar approach was successfully described for peptideantigens of the anti-p24 (HIV-1) monoclonal antibody; see Kramer, Cell91 (1997), 799-809. A general route to fingerprint analyses ofpeptide-antibody interactions using the clustered amino acid peptidelibrary was described in Kramer, Mol. Immunol. 32 (1995), 459-465. Inaddition, antagonists of the inventive lung tumor associated polypeptideof the invention can be derived and identified from monoclonalantibodies that specifically react with the polypeptide of the inventionin accordance with the methods as described in Doring, Mol. Immunol. 31(1994), 1059-1067.

More recently, WO 98/25146 described further methods for screeninglibraries of complexes for compounds having a desired property,especially, the capacity to agonize, bind to, or antagonize apolypeptide or its cellular receptor. The complexes in such librariescomprise a compound under test, a tag recording at least one step insynthesis of the compound, and a tether susceptible to modification by areporter molecule. Modification of the tether is used to signify that acomplex contains a compound having a desired property. The tag can bedecoded to reveal at least one step in the synthesis of such a compound.Other methods for identifying compounds which interact with thepolypeptides according to the invention or nucleic acid moleculesencoding such molecules are, for example, the in vitro screening withthe phage display system as well as filter binding assays or “real time”measuring of interaction using, for example, the BIAcore apparatus(Pharmacia).

All these methods can be used in accordance with the present inventionto identify activators/agonists and inhibitors/antagonists of the lungtumor associated polypeptide or related polypeptide of the invention.

Various sources for the basic structure of such an activator orinhibitor can be employed and comprise, for example, mimetic analoguesof the polypeptide of the invention. Mimetic analogues of thepolypeptide of the invention or biologically active fragments thereofcan be generated by, for example, substituting the amino acids that areexpected to be essential for the biological activity with, e.g.,stereoisomers, i.e. D-amino acids; see e.g., Tsukida, J. Med. Chem. 40(1997), 3534-3541. Furthermore, in case fragments are used for thedesign of biologically active analogs pro-mimetic components can beincorporated into a peptide to re-establish at least some of theconformational properties that may have been lost upon removal of partof the original polypeptide; see, e.g., Nachman, Regul. Pept. 57 (1995),359-370. Furthermore, the lung tumor associated polypeptide of theinvention can be used to identify synthetic chemical peptide mimeticsthat bind to or can function as a ligand, substrate, binding partner orthe receptor of the polypeptide of the invention as effectively as doesthe natural polypeptide; see, e.g., Engleman, J. Clin. Invest. 99(1997), 2284-2292. For example, folding simulations and computerredesign of structural motifs of the polypeptide of the invention can beperformed using appropriate computer programs (Olszewski, Proteins 25(1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679).Computer modelling of protein folding can be used for the conformationaland energetic analysis of detailed peptide and protein models (Monge, J.Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376(1995), 37-45). In particular, the appropriate programs can be used forthe identification of interactive sites of the inventive lung tumorassociated polypeptide and its possible receptor, its ligand or otherinteracting proteins by computer assistant searches for complementarypeptide sequences (Fassina, Immunomethods 5 (1994), 114-120. Furtherappropriate computer systems for the design of protein and peptides aredescribed in the prior art, for example in Berry, Biochem. Soc. Trans.22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987), 1-13;Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from theabove-described computer analysis can be used for, e.g., the preparationof peptide mimetics of the protein of the invention or fragmentsthereof. Such pseudopeptide analogues of the natural amino acid sequenceof the protein may very efficiently mimic the parent protein (Benkirane,J. Biol. Chem. 271 (1996), 33218-33224). For example, incorporation ofeasily available achiral *-amino acid residues into a protein of theinvention or a fragment thereof results in the substitution of amidebonds by polymethylene units of an aliphatic chain, thereby providing aconvenient strategy for constructing a peptide mimetic (Banerjee,Biopolymers 39 (1996), 769-777). Superactive peptidomimetic analogues ofsmall peptide hormones in other systems are described in the prior art(Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327-331). Appropriatepeptide mimetics of the protein of the present invention can also beidentified by the synthesis of peptide mimetic combinatorial librariesthrough successive amide alkylation and testing the resulting compounds,e.g., for their binding and immunological properties. Methods for thegeneration and use of peptidomimetic combinatorial libraries aredescribed in the prior art, for example in Ostresh, Methods inEnzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996),709-715. Furthermore, a three-dimensional and/or crystallographicstructure of the polypeptide of the invention can be used for the designof peptide mimetic inhibitors of the biological activity of thepolypeptide of the invention (Rose, Biochemistry 35 (1996), 12933-12944;Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

The structure-based design and synthesis of low-molecular-weightsynthetic molecules that mimic the activity of the native biologicalpolypeptide is further described in, e.g., Dowd, Nature Biotechnol. 16(1998), 190-195; Kieber-Emmons, Current Opinion Biotechnol. 8 (1997),435-441; Moore, Proc. West Pharmacol. Soc. 40 (1997), 115-119; Mathews,Proc. West Pharmacol. Soc. 40 (1997), 121-125; Mukhija, European J.Biochem. 254 (1998), 433-438.

It is also well known to the person skilled in the art, that it ispossible to design, synthesize and evaluate mimetics of small organiccompounds that, for example, can act as a substrate or ligand to thelung tumor associated polypeptide of the invention or the relatedpolypeptide. For example, it has been described that D-glucose mimeticsof hapalosin exhibited similar efficiency as hapalosin in antagonizingmultidrug resistance assistance-associated protein in cytotoxicity; seeDinh, J. Med. Chem. 41 (1998), 981-987.

The nucleic acid molecule of the invention can also serve as a targetfor activators and inhibitors. Activators may comprise, for example,proteins that bind to the mRNA of a gene encoding a the inventive lungtumor associated polypeptide, thereby stabilizing the nativeconformation of the mRNA and facilitating transcription and/ortranslation, e.g., in like manner as Tat protein acts on HIV-RNA.Furthermore, methods are described in the literature for identifyingnucleic acid molecules such as an RNA fragment that mimics the structureof a defined or undefined target RNA molecule to which a compound bindsinside of a cell resulting in retardation of cell growth or cell death;see, e.g., WO 98/18947 and references cited therein. These nucleic acidmolecules can be used for identifying unknown compounds ofpharmaceutical and/or agricultural interest, and for identifying unknownRNA targets for use in treating a disease. These methods andcompositions can be used in screening for novel antibiotics,bacteriostatics, or modifications thereof or for identifying compoundsuseful to alter expression levels of proteins encoded by a nucleic acidmolecule. Alternatively, for example, the conformational structure ofthe RNA fragment which mimics the binding site can be employed inrational drug design to modify known antibiotics to make them bind moreavidly to the target. One such methodology is nuclear magnetic resonance(NMR), which is useful to identify drug and RNA conformationalstructures. Still other methods are, for example, the drug designmethods as described in WO 95/35367, U.S. Pat. No. 5,322,933, where thecrystal structure of the RNA fragment can be deduced and computerprograms are utilized to design novel binding compounds which can act asantibiotics.

Some genetic changes lead to altered protein conformational states. Forexample, some mutant the inventive lung tumor associated polypeptidesmay possess a tertiary structure that renders them far less capable ofprotein degradation. Restoring the normal or regulated conformation ofmutated proteins is the most elegant and specific means to correct thesemolecular defects, although it may be difficult. Pharmacologicalmanipulations thus may aim at restoration of wild-type conformation ofthe inventive lung tumor associated polypeptide. Thus, the nucleic acidmolecules and encoded polypeptides of the present invention may also beused to design and/or identify molecules which are capable of activatingthe wild-type function of a the inventive lung tumor associatedpolypeptide or related polypeptide.

The compounds which can be tested and identified according to a methodof the invention may be expression libraries, e.g., cDNA expressionlibraries, peptides, proteins, nucleic acids, antibodies, small organiccompounds, hormones, peptidomimetics, PNAs or the like (Milner, NatureMedicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell79 (1994), 193-198 and references cited supra). Furthermore, genesencoding a putative regulator of the inventive lung tumor associatedpolypeptide and/or which exert their effects up- or downstream theinventive lung tumor associated polypeptide may be identified using, forexample, insertion mutagenesis using, for example, gene targetingvectors known in the art. Said compounds can also be functionalderivatives or analogues of known inhibitors or activators. Such usefulcompounds can be for example transacting factors which bind to theinventive lung tumor associated polypeptide or regulatory sequences ofthe gene encoding it. Identification of transacting factors can becarried out using standard methods in the art (see, e.g., Sambrook,supra, and Ausubel, supra). To determine whether a protein binds to theprotein itself or regulatory sequences, standard native gel-shiftanalyses can be carried out. In order to identify a transacting factorwhich binds to the protein or regulatory sequence, the protein orregulatory sequence can be used as an affinity reagent in standardprotein purification methods, or as a probe for screening an expressionlibrary. The identification of nucleic acid molecules which encodepolypeptides which interact with the inventive lung tumor associatedpolypeptides described above can also be achieved, for example, asdescribed in Scofield (Science 274 (1996), 2063-2065) by use of theso-called yeast “two-hybrid system”. In this system the polypeptideencoded by a nucleic acid molecule according to the invention or asmaller part thereof is linked to the DNA-binding domain of the GAL4transcription factor. A yeast strain expressing this fusion polypeptideand comprising a lacZ reporter gene driven by an appropriate promoter,which is recognized by the GAL4 transcription factor, is transformedwith a library of cDNAs which will express plant proteins or peptidesthereof fused to an activation domain. Thus, if a peptide encoded by oneof the cDNAs is able to interact with the fusion peptide comprising apeptide of an inventive lung tumor associated polypeptide, the complexis able to direct expression of the reporter gene. In this way thenucleic acid molecules according to the invention and the encodedpeptide can be used to identify peptides and proteins interacting withthe inventive lung tumor associated protein. It is apparent to theperson skilled in the art that this and similar systems may then furtherbe exploited for the identification of inhibitors of the binding of theinventive lung tumor associated proteins.

Once the transacting factor is identified, modulation of its binding toor regulation of expression of the inventive lung tumor associatedpolypeptide can be pursued, beginning with, for example, screening forinhibitors against the binding of the transacting factor to the proteinof the present invention. Activation or repression of the inventive lungtumor associated proteins could then be achieved in animals by applyingthe transacting factor (or its inhibitor) or the gene encoding it, e.g.in an expression vector. In addition, if the active form of thetransacting factor is a dimer, dominant-negative mutants of thetransacting factor could be made in order to inhibit its activity.Furthermore, upon identification of the transacting factor, furthercomponents in the pathway leading to activation (e.g. signaltransduction) or repression of a gene involved in the control of theinventive lung tumor associated polypeptide then can be identified.Modulation of the activities of these components can then be pursued, inorder to develop additional drugs and methods for modulating themetabolism of protein degradation in animals. Thus, the presentinvention also relates to the use of the two-hybrid system as definedabove for the identification of the inventive lung tumor associatedpolypeptide or activators or inhibitors of the inventive lung tumorassociated polypeptide.

The compounds isolated by the above methods also serve as lead compoundsfor the development of analogue compounds. The analogues should have astabilized electronic configuration and molecular conformation thatallows key functional groups to be presented to the inventive lung tumorassociated polypeptide or its possible receptor in substantially thesame way as the lead compound. In particular, the analogue compoundshave spatial electronic properties which are comparable to the bindingregion, but can be smaller molecules than the lead compound, frequentlyhaving a molecular weight below about 2 kD and preferably below about 1kD. Identification of analogue compounds can be performed through use oftechniques such as self-consistent field (SCF) analysis, configurationinteraction (CI) analysis, and normal mode dynamics analysis. Computerprograms for implementing these techniques are available; e.g., Rein,Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss,New York, 1989). Methods for the preparation of chemical derivatives andanalogues are well known to those skilled in the art and are describedin, for example, Beilstein, Handbook of Organic Chemistry, Springeredition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. andOrganic Synthesis, Wiley, New York, USA. Furthermore, said derivativesand analogues can be tested for their effects according to methods knownin the art; see also supra. Furthermore, peptidomimetics and/or computeraided design of appropriate derivatives and analogues can be used, forexample, according to the methods described above.

In a preferred embodiment of the above-described methods of theinvention, said cell is a cell of or, obtained by a method of theinvention or is comprised in the above-described transgenic non-humananimal.

Once the described compound has been identified and obtained, it ispreferably provided in a therapeutically acceptable form.

The present invention provides methods for detection and treatment ofdisorders characterized by abnormal cell proliferation, such as e.g.cancers. In one aspect the present invention provides a method for thedetection of disorders characterized by abnormal cell proliferation,such as e.g. cancers based on the determination of the presence orabsence and/or the level of expression of the inventive lung tumorassociated gene in biological samples. In a second aspect the presentinvention provides a method for treatment of disorders characterized byabnormal cell proliferation, such as e.g. cancers using the inventivelung tumor associated gene products as therapeutically active agents.The invention also provides for therapeutic methods based on themodulation of the activity of the inventive lung tumor associatedpolypeptide. It is one aspect of the invention to provide a method forrational tumor management based on the detection of the inventive lungtumor associated gene products in patient samples and the tailoring of atherapy correlated to the detected overexpression of said gene products.Furthermore the present invention provides for a research or diagnostictest kit for performing the reactions involved in the detection of thepresence or absence and/or the level of overexpression of the inventivelung tumor associated gene. Finally the present invention relates topharmaceutical compositions applicable in the treatment of disordersaccording to the present invention.

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the invention disclosedherein.

EXAMPLES Example 1 Real-Time RT-PCR Analysis of LUMA1 Expression inTumor Samples Including Colon Carcinoma, Carcinoma of the Stomach, SmallCell Lung Cancer and Lung Adenocarcinoma

An upregulation of LUMA1 transcripts has been detected in 60% of testedlung adenocarcinomas, whereas no upregulation was found in coloncarcinoma, carcinoma of the stomach and small cell lung cancer.

1.1. Theoretical Basis

Quantitative values were obtained from the threshold cycle number atwhich the increase in the signal associated with an exponential growthof PCR products started to be detected (using PE Biosystems analysissoftware), according to the manufacturer's manuals.

6.25 ng cDNA of oligo dT primed total RNA was added to each real timePCR. We quantified transcripts of the beta-actin gene (ACTB Actin,Primer Actin1—CCTAAAAGCCACCCCACTTCTC (SEQ ID NO: 28), PrimerActin2—ATGCTATCACCTCCCCTGTGTG (SEQ ID NO: 29)) encoding human actin,beta (ACTB) as the endogenous RNA control.

Final results, expressed as N-fold differences in target gene expressionrelative to the reference gene ACTB, termed ‘Ntarget’, were determinedas follows:N _(target)=2^((delta Ct) ^(sample) ^(−delta Ct) ^(reference gene) ⁾where delta Ct values of the sample and reference were determined bysubtracting the average Ct value of the WT1 gene from the average Ctvalue of the ACTB gene.

Primers for WT1 and ACTB gene were chosen with the assistance of thecomputer programs PRIMER (Husar program package, DKFZ Heidelberg) andPrimer Express (Perkin-Elmer Applied Biosystems, Foster City, Calif.).The following nucleotide sequences of primers for the amplification ofthe LUMA1 gene were used: LUMA1-A: CTCGTCAGGCGACCTTATATC (SEQ ID NO: 24)and LUMA1-B: TGTCAGTTGAACATTTTCTGCC (SEQ ID NO: 25); LUMA1-C:CTCGTCAGGCGATACTCCC (SEQ ID NO: 26) and LUMA1-D: CACCAGTCAGCTCTAAATGGG(SEQ ID NO: 27).

1.2. RNA Extraction

Total RNA was extracted from tissue specimens by using the QIAamp RNAMini Protocol (Qiagen, Hilden, Germany).

1.3. cDNA Synthesis

1 μg total RNA was DNAse I digested for 15 min at 25° C. in a finalvolume of 20 μl containing 1 μl DNAse I Amp Grade (1 Unit/μl;Invitrogen) and 2 μl DNAse Reaction Buffer (10×; Invitrogen). Thereaction was stopped by adding 2 μl EDTA (25 mM; Invitrogen) andincubation for 10 min at 65° C. The reverse transcription was performed2 h at 37° C. in a final volume of 40 μl containing 4 μl 10× RT buffer,4 μl 5 mM dNTP, 1 μl RNAsin 40 U/μl Promega), 4 μl Oligo dT Primer 0.5μg/ml and 2 μl Omniscript 4 U/μl (Qiagen, Hilden, Germany). Reversetranscriptase was inactivated by heating at 93° C. for 5 min and coolingat 4° C. for 5 min.

1.4. PCR Amplification

All PCR reactions were performed using a ABI Prism 7700 SequenceDetection System (Perkin-Elmer Applied Biosystems). PCR was performedusing the SYBR® Green PCR Mix (Perkin-Elmer Applied Biosystems). Thethermal cycling conditions comprised an initial denaturation step at 95°C. for 10 min and 40 cycles at 95° C. for 15 s and 60° C. for 1 min.Experiments were performed with duplicates for each data point.

As shown in FIG. 16-20, an enhanced expression of LUMA1 transcripts inlung adenocarcinomas has been detected. For amplification of the LUMA1transcripts the following primers were used: LUMA1-A:CTCGTCAGGCGACCTTATATC (SEQ ID NO: 24) and LUMA1-B:TGTCAGTTGAACATTTTCTGCC (SEQ ID NO: 25). The PCR with primer LUMA1-A andLUMA1-B amplified the splice variant which harboured the complete exon7, whereas the primers LUMA1-C (CTCGTCAGGCGATACTCCC, SEQ ID NO: 26) andLUMA1-D (CACCAGTCAGCTCTAAATGGG, SEQ ID NO: 27) amplified the splicevariant, which represented only part of exon 7. Using these primercombinations, an upregulation of both splice variants was observed inreal time PCR experiments. In FIG. 16 the real time amplification oftumor 1 and corresponding normal sample 1 is shown (primer LUMA1-A andLUMA1-B). A threshold cycle difference of three was observed whereaswith the reference primers for the ACTB gene no difference was detected(not shown). This indicates a 8-fold overexpression of the LUMA1transcript in lung adenocarcinoma of one individual. In anotherindividual, no differences between normal and lung adenocarcinoma tissuewas observed (FIG. 14). One further individual showed a 13-foldoverexpression in the tumor tissue (FIG. 15). More individuals werecharacterized by the absence of LUMA1 transcript in normal tissue and anstrong upregulation in the tumor tissue (more than 4000-fold) (FIG. 16;17). The PCR products of the two different analysed exon 7 variantsshown in FIG. 23 were obtained by the Real time PCR described above andwere analyzed by gel electrophoresis.

Example 2 Cloning of Differentially Spliced LUMA1 Transcripts

Using primers specific for the LUMA1, transcript PCR was performed withrandom primed human cDNA derived from lung adenocarcinoma, coloncarcinoma and fetal brain.

Forward primers: LUMA1-C: CTCGTCAGGCGATACTCCC; LUMA1-E:ATGGAAATCACCACTCTGAGAG; (SEQ ID NO: 30) LUMA1-F: TTGATCCAGAAAGTGTGTGAGC(SEQ ID NO: 31) Reverse primers: LUMA1-G: TGCAGTTGGCCCAGCTTAGAA; (SEQ IDNO: 32) LUMA1-H: AGTCTTTAAAAAGCGTTGCTGG (SEQ ID NO: 33)

The following primer combinations were used to amplify:

LUMA1-C+LUMA1-H; LUMA1-E+LUMA1-G; LUMA1-E+LUMA1-H; LUMA1-F+LUMA1-G;LUMA1-F+LUMA1-H

The following PCR conditions used were to amplify the differentiallyspliced LUMA1 transcripts:

95° C. 1 min, (95° C. 30 sec, 60° C. 1 min, 68° C. 3.5 min)-34 cycles

The TaqAdvantage polymerase (Clontech) was used.

1 μl dNTP (20 mM), 0.5 μl 50× TaqAdvantageII (Clontech), 2.5 μl 10×Buffer, 3 μl cDNA (100 ng), 1.5 μl (Primer forward, 10 μm), 1.5 μl(Primer reverse, 10 μm), 15 μl H₂O.

The PCR products were directly sequenced or first cloned into the pCR2.1vector (Invitrogen) and then sequenced. Sequence analysis and databasesearches were performed with the HUSAR program package (DKFZHeidelberg). Sequence analysis of the cloned PCR products hasdemonstrated an extensive splicing of the LUMA1 transcript. Onetranscript has been identified which was characterized by the absence ofexon 3. Another transcript missed exon 3 and 4. In addition, atranscript has been cloned where exon 3, 4 and 5 was spliced out.Splicing out the exons 3 or 4 or 5 or combinations thereof do not changethe reading frame and lead to internal deletions in the encoded LUMA1protein isoforms. In contrast to these splice variants, the differentialsplicing at exon 7 led to a frameshift. If the complete exon 7 waspresent in the transcript, the open reading frame almost extended to the3′ end of the transcript. If the first part of exon 7 was spliced out inthe resulting transcript a frameshift was generated which lead to ashorter carboxy terminus of the encoded protein. Both exon 7 splicevariants were shown to be upregulated in lung adenocarcinomas (FIG.13-17; FIG. 18-19).

Example 3 Full Length Cloning of LUMA1

Full length cloning of LUMA1 (Rapid Amplification of cDNA Ends)

Full length cloning was performed using SMART™ RACE cDNA AmplificationKit (Clontech) to amplify the 5′ end of LUMA1 cDNA.

PCR reaction was prepared as follows: 1 μl dNTP (10 mM), 1 μl 50×TaqAdvantageII (Clontech), 5 μl 10× Buffer, 2.5 μl cDNA, placenta(Clontech) (1 μg/μl), 5 μl Universal Primer Mix (Clontech)(10×), 1 μlGene specific Primer, reverse (Clontech), 34.5 μl H₂O.

Universal Primer Mix: SEQ ID NO: 34CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT, (0.4 μm), SEQ ID NO: 35CTAATACGACTCACTATAGGGC, (0.2 μm) LUMA1 Gene specific Primer, reverse:(SEQ ID NO: 36) GGAGATGGAGCTGTTTGACCTGA

Because PCR reaction failed to give a distinct band, nested PCR wasperformed.

5 μl of primary PCR product were diluted into 245 μl Tricine-EDTA buffer(Clontech) (10 mM Tricine-KOH (pH 8.5), 1 mM EDTA)

PCR reaction was prepared as follows: 1 μl dNTP (10 mM), 1 μl 50×TaqAdvantageII (Clontech), 5 μl 10× Buffer, 5 μl diluted primary PCRproduct, 1 μl Nested Universal Primer (Clontech)(10×), 1 μl nested Genespecific Primer, reverse (Clontech)(10 μm), 36 μl H₂O.

Nested Universal Primer: AAGCAGTGGTATCAACGCAGAGT, SEQ ID NO: 37 LUMA1nested Gene specific Primer, reverse: TGTCAGTTGAACATTTTCTGCC, SEQ ID NO:38

The following PCR conditions used were to generate full lengthtranscripts:

(94° C. 30 sec 72° C. 3 min)-5 cycles

(94° C. 30 sec, 70° C. 30 sec, 72° C. 3 min)-5 cycles

(94° C. 30 sec, 68° C. 30 sec, 72° C. 3 min)-27 cycles

RACE product was detected in agarose gel electrophoresis. Bands weregel-purified using the High Pure PCR Product Purification Kit (Roche).DNA band was cut from agarose gel (1%) using an ethanol-cleaned scalpel.Agarose gel slice was placed in a tube and transferred for 2 min intoliquid nitrogen. The gel slide was placed into a sterile filtermicrocentrifuge tube and centrifuged for 10 min at 1300 rpm. 500 μlBinding buffer was added to every 100 μl flowthrough and placed in a newsterile filter microcentrifuge tube and centrifuged for 1 min at 1300rpm Sample was washed two times with 500 μl Washing Buffer andcentrifuged for 1 min at 1300 rpm. Flowthrough was discarded. 50 μlElution Buffer was added to the upper reservoir of the filter tube andcentrifuged for 1 min at 1300 rpm. The microcentrifuge tube containedthe purified DNA.

Purified DNA was cloned into the PCR-XL-TOPO vector (Invitrogen) andthen sequenced. Sequence analysis and database searches were performedwith the HUSAR program package (DKFZ Heidelberg). Sequence analysis ofthe cloned PCR products identified 12 new exons (A, B, C, D, E, F, G, H,I, J, K, L) and new part of exon1 (exon M) and a 5′ extension of exon 8(exon T).

Additional LUMA1 exons were identified with the RACE experiments. Thelocation of LUMA1 exons are indicated in respect to the genomic cloneAL359711 (VERSION AL359711.18 GI:13234940/Human DNA sequence from cloneRP11-425D10 on chromosome 6, complete sequence) (Acc. No. al359711).

bp Exon A 16742-16898 Exon B 17967-18132 Exon C 22671-22809 exon isdifferentially spliced Exon D 28399-28552 Exon E 30422-32094 Exon F36503-36732 Exon G 37720-37858 Exon H 39288-39408 Exon I 46143-46290differentially spliced, present in fetal lung Exon J 51443-51552 Exon K58808-58957 Exon L 59836-59889 Exon M Exon 1 60888-61055 Exon N Exon 263342-63494 Exon O Exon 3 63739(63742)-63873 alternative start of exonat bp 63742, exon is differentially spliced Exon P Exon 4 64839-64991exon is differentially spliced Exon Q Exon 5 66427-66636 exon isdifferentially spliced Exon R Exon 6 70867-70987 Exon S Exon 775227(75279)-75354 alternative start of exon at bp 75279, both exonvariants are differentially spliced, alternative start of exon leads toa frameshift, complete exon can be spliced out Exon T Exon 876537(77287)-77410 alternative start of exon at bp 77287, alternativestart of exon leads to a frameshift art of exon leads to a frameshift

Example 4 Generation of Antibodies

LUMA1 peptides were used to produce monoclonal as well as polyclonalantibodies directed against these polypeptides. For LUMA exon 2 thefollowing peptide has been used for immunisation immunization (C E L I GE V R T E G I D N M K D L K, SEQ ID NO: 39) to generate polyclonalantibodies. For the generation of monoclonal antibodies, peptidesequences encoded by the LUMA exon 7b (long variant) have been used (R FL K T N L K G S K I T R C, SEQ ID NO: 40).

A. Production of the Polyclonal Antibodies:

The polyclonal antibodies were purified by affinity chromatography andafterwards used for the detection of the respective polypeptides inpatient samples. The procedures were performed as follows:

NZW (New Zealand White) Rabbits were immunized with 100 μg LUMA1peptides coupled to KLH (ground immunization) in Complete FreundsAdjuvants and boosted 4 times with the same amount of protein inincomplete Freunds Adjuvants at intervals of 2 weeks.

Blood was taken one week after the 2^(nd) boost and subjected to ELISAon the immobilized Antigen. One week after the final boost animals weresubjected exsanguination. After coagulation the final blood wascentrifuged at 4000×g for 10 minutes. The supernatant from this step wascentrifuged again at 16600×g for 15 min. The supernatant represented theraw LUMA1 antiserum.

The antiserum was purified in 2 steps. Step 1 represented a conventionalProtein A Chromatography. In step 2, the Ig fraction of step 1 waspurified by Affinity chromatography on the Antigen immobilized ontoSEPHAROSE® (high molecular weight substance for separation by gelfiltration of macromolecules). The eluate of step 2 represented thepurified LUMA1 antiserum.

The purified antiserum was tested in several dilutions 1/1000- 1/1000000 on the immobilized antigen by peptide ELISA. The specific antibodieswere detected by anti Rabbit secondary reagents coupled to horseradishperoxidase (HRP) with a subsequent colorimetric reaction (e.g. TMB).

In a second approach, the purified Antiserum was evaluated by WesternBlot with immobilized Antigen subsequently to SDS-PAGE and transfer ontoNitrocellulose.

In a last evaluation step, the antiserum was tested on tissue arrays byImmuno-histochemistry (IHC). In Western Blot Analysis as well as withIHC bound antibodies were visualized with anti Rabbit secondary Reagentsconjugated to HRP catalysing a colorimetric reaction.

B. Monoclonal Antibodies

Monoclonal antibodies directed against the peptide sequence (R F L K T NL K G S K I T R C, SEQ ID NO: 40) encoded by the LUMA exon 7b (longvariant) were generated as described in Harlow and Lane. Antibodies: ALaboratory Manual, 1^(st) Edition, 1988.

Example 5 Immunohistochemical Detection of Expression of LUMA1

Sections of formalin fixed, paraffin embedded tissue samples of the lungwere immunocytochemically stained using antibodies specific for LUMA1.

The sections were rehydrated through incubation in xylene and gradedethanol, and transferred to Aqua bidest. Antigen Retrieval was carriedout with 10 mM citrate buffer (pH 6.0) Thereafter the slides were heatedin a water bath for 40 min at 95° C. The slides were cooled down to RTfor 20 minutes, transferred to washing buffer (PBS/0.1% TWEEN® 20(polyoxyethylene sorbitan monolauate)).

For inactivation of endogenous peroxidase the samples were incubatedwith 3% H2O2 for 20 min at RT and afterwards washed in PBS/0.1% TWEEN®20 for 5 to 10 min. The slides were then incubated with the primaryantibodies specific for exon 2 (FIG. 24) and exon 7 (FIG. 25, 26)respectively (2 μg/ml) (for 1 hour at RT, the slides were then rinsedwith washing buffer and placed in a fresh buffer bath for 5 min.

Afterwards, the slides were incubated with the secondary antibody (goatanti rabbit (1:500) or goat anti mouse (1:500) for 1 hour at RT. Washingwas performed 3 times for 5 minutes. Slides were covered with 200 μlsubstrate-chromogen solution (DAB) for 10 min. Then slides were washedas before and counterstained for 2 min in a bath of haematoxylin.Residual haematoxylin was rinsed with distilled water, and specimenswere mounted and coverslipped with an aqueous mounting medium.

The microscopic examination of the slides revealed that tumor cellsshowed a specific immunoreactivity with the monoclonal LUMA1 antibodydirected against exon 7 encoded sequences. In the tumor cells, aspecific staining was visible in the cytoplasm.

Immunochemical analysis of peripheral venous blood, of bone marrow andof lymphocytes by the described methods revealed no immunoreactivity forLUMA1 in samples obtained from normal control individuals. Thisindicates that disseminated tumour cells that are immunoreactive withLUMA1 can be identified in these samples by specific immunochemicalstaining with antibodies directed against LUMA1.

Summary of Immunohistochemical Analysis of Lung Tumor Tissues andCorresponding Normal Tissues Employing a Monoclonal Antibody DirectedAgainst LUMA1 Exon 7.

14 lung tumors have been analysed for the expression of the LUMA exon 7protein isoform. 8 tumors showed a positive staining. The correspondingnormal tissues did not show a staining. In the connective tissue of somelung tumors, a staining of inflammatory cells was detected. The resultsshow that the staining with reagents specific for LUMA1 exon 7 sequencesallowed to identify tumor cells in biological samples. The resultsobtained at the protein level did correlate with the results from theReal time PCR. In both experiments, the LUMA1 exon 7 sequences (RNAsequences and protein sequences) were found to be specificallyupregulated in tumor cells. In contrast to the exon 7 sequences, theexon 2 of LUMA did not show an upregulation either at the RNA or theprotein level in tumor cells. This clearly indicates that only specificLUMA1 splice variants and encoded protein variants are tumor specific.

1. A method for detecting lung adenocarcinoma, squamous cell lungcarcinoma, clear cell lung carcinoma or non-small cell lung carcinoma inan individual comprising: a. obtaining a lung tissue sample from anindividual, b. determining the level of a nucleic acid comprising asequence that encodes the amino acid sequence of SEQ ID NO:2 in saidlung tissue sample, c. comparing the level of said nucleic acid in saidlung tissue sample to the level of said nucleic acid determined in anon-cancerous lung tissue sample, and d. determining that there is anindication of the presence of lung adenocarcinoma, squamous cell lungcarcinoma, clear cell lung carcinoma or non-small cell lung carcinoma inthe individual if the level of said nucleic acid is higher in the lungtissue sample from said individual than the level of said nucleic acidin the non-cancerous lung tissue sample.
 2. The method according toclaim 1, wherein the level of said nucleic acid is determined using anucleic acid probe or primer.
 3. The method according to claim 2,wherein the nucleic acid probe is detectably labeled.
 4. The methodaccording to claim 3, wherein the label is selected from the groupconsisting of a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate,biotin, digoxygenin or an enzyme.
 5. The method according to claim 2,wherein the determination of the level of said nucleic acid comprises anucleic acid amplification reaction.
 6. The method according to claim 2,wherein the determination of the level of said nucleic acid is performedby in-situ detection.
 7. The method according to claim 1, which iscarried out in the course of an in vitro molecular imaging method. 8.The method according to claim 1, wherein said nucleic acid comprises thenucleic acid sequence of SEQ ID NO:
 1. 9. A method for detecting lungadenocarcinoma, squamous cell lung carcinoma, clear cell lung carcinomaor non-small cell lung carcinoma in an individual comprising: a.obtaining a lung tissue sample from an individual, b. determining thelevel of a nucleic acid comprising a sequence that encodes the aminoacid sequence of SEQ ID NO:6 in said lung tissue sample, c. comparingthe level of said nucleic acid in said lung tissue sample to the levelof said nucleic acid determined in a non-cancerous lung tissue sample,and d. determining that there is an indication of the presence of lungadenocarcinoma, squamous cell lung carcinoma, clear cell lung carcinomaor non-small cell lung in the individual if the level of said nucleicacid is higher in the lung tissue sample from said individual than thelevel of said nucleic acid in the non-cancerous lung tissue sample. 10.The method according to claim 9, wherein the level of said nucleic acidis determined using a nucleic acid probe or primer.
 11. The methodaccording to claim 10, wherein the nucleic acid probe is detectablylabeled.
 12. The method according to claim 11, wherein the label isselected from the group consisting of a radioisotope, a bioluminescentcompound, a chemiluminescent compound, a fluorescent compound, a metalchelate, biotin, digoxygenin or an enzyme.
 13. The method according toclaim 10, wherein the determination of the level of said nucleic acidcomprises a nucleic acid amplification reaction.
 14. The methodaccording to claim 10, wherein the determination of the level of saidnucleic acid is performed by in-situ detection.
 15. The method accordingto claim 9, which is carried out in the course of an in vitro molecularimaging method.
 16. The method according to claim 9, wherein saidnucleic acid comprises the nucleic acid sequence of SEQ ID NO: 5.